Radiolabeled alpha-melanocyte stimulating hormone hybrid peptide for melanoma targeting

ABSTRACT

The present invention is directed to novel non-invasive diagnostic tools/compounds to image cancers, especially, melanoma, including metastatic melanoma in vivo. The present compounds exhibit enhanced uptake in cancerous cells and tissue, suggesting favorable selective activity of compounds according to the present invention, which can be used as effective therapeutic agents against melanoma, including metastatic melanoma. The compounds according to the present invention represent an advance in the diagnosis and treatment of melanoma, including metastatic melanoma using non-invasive molecular imaging techniques. The novel probes of the present invention are useful to initiate therapy for melanoma as well as monitor patients&#39; response to chemotherapy treatments and other interventions or therapies used in the treatment of melanoma/metastatic melanoma. Compounds according to the present invention may be used as diagnostic and therapeutic tools for a number of conditions and diseases states, especially melanoma.

This application claims the benefit of priority of United Statesprovisional application serial number U.S. 61/540,814, filed Sep. 29,2011, which application is incorporated by reference in its entiretyherein.

RELATED APPLICATIONS AND GOVERNMENT SUPPORT

The present invention was made with Government support under grant no.NM-INBRE P20RR016480 from the United States NIH. Consequently, the U.S.government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to novel non-invasive diagnostictools/compounds to image cancers, especially, melanoma, includingmetastatic melanoma in vivo. The present compounds exhibit enhanceduptake in cancerous cells and tissue, suggesting favorable selectiveactivity of compounds according to the present invention. The compoundsaccording to the present invention represent an advance in the diagnosisand treatment of melanoma, including metastatic melanoma usingnon-invasive molecular imaging techniques. The novel probes of thepresent invention will also be useful to initiate therapy for melanomaas well as monitor patients' response to chemotherapy treatments andother interventions or therapies used in the treatment ofmelanoma/metastatic melanoma. Compounds according to the presentinvention may be used as diagnostic and therapeutic tools for a numberof conditions and diseases states, especially melanoma.

BACKGROUND OF THE INVENTION

Melanoma is the most deadly skin cancer with an increasing incidence[1]. Over the past decade, it has been of interest to developreceptor-targeting radiolabeled peptides for melanoma imaging sinceearly diagnosis followed by prompt surgical removal is a patient's bestopportunity for a cure. Due to the over-expression on melanoma, bothmelanocotin-1 (MC1) and α_(v)β₃ integrin receptors have been used astargets for radiolabeled alpha-melanocyte stimulating hormone (α-MSH)[2-14] and Arg-Gly-Asp (RGD) peptides [15-22], respectively. Recently,the inventors have developed a novel RGD-conjugated α-MSH hybrid peptidetargeting both MC1 and α_(v)β₃ integrin receptors for M21 human melanomaimaging [23]. The cyclic RGD {Arg-Gly-Asp-DTyr-Asp} motif was conjugatedto [Cys^(3,4,10), D-Phe⁷, Arg¹¹]α-MSH₃₋₁₃} peptide via a lysine linkerto yield RGD-Lys-(Arg¹¹)CCMSH peptide. Meanwhile, the inventors designedtwo control peptides namely RAD-Lys-(Arg¹¹)CCMSH andRGD-Lys-(Arg¹¹)CCMSH scramble for comparison. The in vitro resultsrevealed that the switch from RGD to RAD in the hybrid peptide decreasedthe α_(v)β₃ integrin receptor binding affinity by 248-fold, whereas thescramble of CCMSH moiety in the hybrid peptide sacrificed the MC1receptor binding affinity by 100-fold. The biodistribution resultsdemonstrated that targeting both MC1 and α_(v)β₃ integrin receptorsenhanced the melanoma uptake of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH in M21human melanoma xenografts. The xenografted M21 human melanoma lesionswere clearly visualized using ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH as animaging probe [23].

While the switch from RGD to RAD in the hybrid peptide decreased theα_(v)β₃ integrin receptor binding affinity by 248-fold, surprisingly,the inventors found that the switch from RGD to RAD in the hybridpeptide dramatically increased the MC1 receptor binding affinity ofRAD-Lys-(Arg¹¹)CCMSH compared to RGD-Lys-(Arg¹¹)CCMSH (0.3 vs. 2.0 nM)in M21 melanoma cells [23]. Therefore, the inventors were interested ininvestigating whether such change in MC1 receptor binding affinity couldresult in enhanced melanoma uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHcompared to ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH. Thus, the inventors examinedthe receptor binding affinity of RAD-Lys-(Arg¹¹)CCMSH, internalizationand efflux properties of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH in B16/F1melanoma cells. Furthermore, the inventors determined the melanomatargeting and imaging properties of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH inB16/F1 melanoma-bearing C57 mice. The results proved to be unexpectedlyfavorable to prior art compounds.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to compounds according to the generalstructure:

Where Q is an amino acid unit selected from the group consisting ofglutamic acid and aspartic acid, preferably glutamic acid;R is an amino acid unit selected from the group consisting of valine,threonine, leucine, and isoleucine, preferably valine;V is an amino acid residue selected from the group consisting ofaspartic acid and glutamic acid, preferably aspartic acid;W is an amino acid selected from the group consisting of aspartic acidand glutamic acid, preferably aspartic acid;X is an amino acid residue selected from the group consisting ofalanine, valine threonine, leucine, isoleucine, serine, aspartic acidand glutamic acid, preferably alanine;Y is an amino acid residue selected from the group consisting ofarginine, lysine, alanine, valine, threonine, leucine, isoleucine,serine, aspartic acid and glutamic acid, preferably arginine;L is absent, a single amino acid selected from the group consisting ofglycine, alanine, β-alanine, lysine and arginine, or a linker groupaccording to the formula:

Where each X¹ is independently an amino acid residue (preferably, forexample, an amino acid group which is neutral (e.g. a neutral amino acidsuch as norleucine (Nle), leucine, isoleucine, glycine or alanine) or ispositively charged at physiological pH (arginine, lysine) and ispreferably selected from the group consisting of glycine, alanine,arginine or lysine, or is an amino acid linker comprising an alkylenegroup which is optionally substituted with one or more C₁-C₃ alkyl orC₁-C₃ alkanol group(s) or an ethylene glycol containing group accordingto the chemical structures:

Where ABC is an amino acid linker wherein A is absent or is a neutral orpositively charged amino acid at physiological pH;B is a neutral or positively charged amino acid at physiological pH;C is absent or is a neutral or negatively charged amino acid atphysiological pH;m is an integer from 0 to 250, from 1 to 100, often 0 to 5, preferably 0or 1;each n is independently 0 or 1, preferably at least one n is 0 when m is1 or more;p is an integer from 0 to 20, often 0 to 10, often 1-8, often 2-5;k is an integer from 0 to 10, preferably 1 or 2;i is an integer from 0 to 10, often 1 or 2;s is an integer from 0 to 10, often 0, 1 or 2, more often 0; andM is a radioisotope, preferably a polyvalent cationic radioisotope, evenmore preferably selected from the group consisting of ⁸⁶Y, ⁹⁰Y, ¹¹¹In,¹⁷⁷Lu, ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶⁷Cu, ⁷¹As, ⁷²As,⁷⁶As, ⁷⁷As, ⁶⁵Zn, ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl,¹⁸⁸Re, ¹⁸⁶Re, and ^(99m)Tc, ora pharmaceutically acceptable salt thereof.Preferred compounds according to the present invention are representedby the chemical formula:

Where Q is glutamic acid;R is valine;V is aspartic acid;W is aspartic acid;X is alanine;Y is arginine;L is absent, a single amino acid selected from the group consisting ofglycine, alanine, β-alanine, lysine and arginine, or a

group,Where p is an integer 0-6, often 1-5;k is an integer from 0 to 20, often 1-5, more often 1 or 2;i is an integer from 0 to 10, often 0, 1 or 2, more often 0;s is an integer from 0 to 10, often 0, 1 or 2, more often 0; and

M is ^(99m)Tc or

a pharmaceutically acceptable salt thereof.

Radioisotopes are selected based on the physical half life, the decaymode (alpha, beta, auger, gamma, X-ray) and the energy of theradioisotope. In diagnostic aspects of the present invention, preferredradioisotopes include, for example, ¹¹¹In, ⁸⁶Y, ⁸⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ²⁰³Pb,⁶⁴Cu and ^(99m)Tc.

Where compounds are to be analyzed using positron emission tomography orPET imaging they are labeled with a positron emitting radioisotopes suchas: ⁶⁶Ga, ⁶⁸Ga, ⁶⁴Cu, ⁸⁶Y, or other polyvalent, cationic radiometalsthat decay by positron emission. In alternative embodiments, thecompounds may be analyzed using single photon emission computedtomography or SPECT imaging when labeled with a gamma radiation emittingradioisotope which preferably includes ¹¹¹In, ⁶⁷Ga, ^(99m)Tc and ²⁰³Pbor other gamma emitting radioisotope as disclosed herein.

The present invention relates to compounds and/or compositions which maybe used to prepare imaging/therapeutic agents or as imaging/therapeuticagents (when complexed with a radioisotope) for diagnosing and treatingmelanoma, including metastatic melanoma as otherwise described herein.Compounds according to the present invention which are complexed with anappropriate radioisotope may be used to diagnose the existence and/orextent of melanoma, including metastatic melanoma, monitor therapy as atherapeutic aid of melanoma, including metastatic melanoma, and incertain instances, function as a therapeutic agent (peptide targetedradiation) for the treatment of melanoma, including metastatic melanoma.

The present invention also relates to pharmaceutical compositionscomprising an effective amount of a compound according to the presentinvention which has been complexed with a radioisotope and combined witha carrier, additive or excipient in pharmaceutical dosage form as adiagnostic imaging agent or as a therapeutic agent. Compositionsaccording to the present invention are formulated in pharmaceuticaldosage form for administration preferably by a parenteral, preferably anintravenous route. Compositions according to the present invention mayalso be formulated for administration via a topical route, directly tothe skin. Oral compositions may also be formulated for use in thepresent invention.

In the diagnostic method according to the present invention, a compoundaccording to the present invention is administered to a patient, andevidence of elevated expression of MSH (especially MC1) receptors intissue of said patient through standard well-known nuclear imagingtechniques, especially radiation (radionuclide) imaging, includingscintigraphic imaging, and especially single photon emission computedtomography (SPECT) and positron emission tomography (PET) in comparisonto a normal standard, is indicative of a disease state (melanoma) andextent of disease state (metastasis) in the tissue of the patient. Thenuclear imaging techniques useful in the present diagnostic methods arewell known in the art. In general, elevated levels of radiationemanating from a diagnosed tissue is evidence of elevated MSH (includingMC1) receptor activity and indicative of a disease state or condition(melanoma and/or metastatic melanoma) wherein these receptors are foundat elevated levels. Methods of diagnosing the existence and/or extent(stage) of melanoma, including metastatic melanoma are thereforeadditional aspects of the present invention. Thus, a diagnostic methodof diagnosing the existence or absence of melanoma in a patient at riskfor melanoma comprises administering to said patient a compoundaccording to the present invention; imaging said patient to determine iftissue in said patient exhibits elevated expression of MSH (especiallyMC1) receptors; and diagnosing said patient as having melanoma,including metastatic melanoma if said tissue evidences elevatedexpression of MSH (especially MC1) receptors in comparison to astandard.

Methods of monitoring the treatment of melanoma, including metastaticmelanoma in conjunction with traditional or experimental melanomatherapy is an additional aspect of the invention. In this aspect, apatient's response to therapy is monitored using the methods accordingto the present invention. In this method, a patient is monitored beforeand after therapy by administering compound according to the presentinvention and determining (through imaging diagnostics as otherwisedescribed herein) the extent of expression of melanocyte stimulatinghormone receptors (especially MC1 receptors) in tissues of a patientbefore therapy and after therapy and comparing the expression levelswith each other and/or with a standard (predetermined value) todetermine the extent of reduction of cancer tissue which occurredpursuant to the therapeutic intervention.

Methods of treating melanoma represent a further aspect of theinvention. In this aspect, compounds according to the present inventionas described above are administered to a patient known to have melanomaand/or metastatic melanoma in effective amounts in order to reducecancer tissue and otherwise treat the patient's cancer through targetedradiation therapy. The present therapeutic methods may be used alone orin combination with other treatment methods (surgery, chemotherapy,radiation therapy and/or immunotherapy (IL-2 and α-interferon) formelanoma/metastatic melanoma as otherwise disclosed herein. In preferredtherapeutic method aspects of the present invention, compounds accordingto the present invention are labeled with ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,²¹²Bi/²¹²Pb, ²¹³Bi, ¹⁴⁹Pm, ¹⁶⁶Ho and ¹⁵³Sm, often ¹⁸⁶Re and ¹⁸⁸Re, andare administered to the patient (preferably intravenously ortopically—i.e, directly onto the melanoma tissue in the skin of thepatient) in order to target the malignant melanoma tumor, includingmetastatic melanoma tissue with radiation therapy. Compounds accordingto the present invention, because of their selective binding to MC1receptors and their residence in melanoma cells provide for enhanceddiagnostic and therapeutic methods over compounds which are known in theart.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the schematic structures of RAD-Lys-(Arg¹¹)CCMSH andRGD-Lys-(Arg¹¹)CCMSH.

FIG. 2 shows the competitive binding curve of RAD-Lys-(Arg¹¹)CCMSH inB16/F1 melanoma cells. The IC₅₀ value of RAD-Lys-(Arg¹¹)CCMSH was 0.26nM.

FIG. 3 shows the cellular internalization (A) and efflux (B) of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH in B16/F1 melanoma cells. Total boundradioactivity (♦), internalized radioactivity (▪) and cell membraneradioactivity (▴) were presented as counts per minute (cpm).

FIG. 4 shows the whole-body (A), coronal (B) and transversal (C)SPECT/CT images of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH in a B16/F1melanoma-bearing C57 mouse at 2 h post-injection.

FIG. 5 shows the radioactive HPLC profile of urine sample of a B16/F1melanoma-bearing C57 mouse at 2 h post-injection of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH. Arrow indicates the retention time (12.6min) of the original compound of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH prior tothe tail vein injection.

FIG. 6 shows the comparison in tumor, liver and kidney uptake between^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH and ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH. Thedata of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH was cited from ref. 24.

FIG. 7, Table 1 shows the biodistribution of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice. Thedata was presented as percent injected dose/gram or as percent injecteddose (mean±SD, n=5)

DETAILED DESCRIPTION OF THE INVENTION

The following terms are used to describe the present invention. In theevent that a term is not specifically defined herein, that term isaccorded its commonly understood meaning within the context of its useby those of ordinary skill in the art. It is understood that thedefinitions of the terms which are used to describe the presentinvention are interpreted in a manner consistent with the presentinvention and within the context of a particular term's use indescribing the present invention in one or more embodiments.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a compound”, withincontext, includes a plurality (for example, two or more compounds) ofsuch elements, and so forth. Under no circumstances is the patent beinterpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein.

The term “patient” or “subject” is used throughout the specification todescribe an animal, preferably a human, to whom treatment, includingprophylactic treatment, with the compounds according to the presentinvention is provided. For treatment of those infections, conditions ordisease states which are specific for a specific animal such as a humanpatient, the term patient refers to that specific animal.

The term “compound” is used herein to refer to any specific chemicalcompound disclosed herein. Within its use in context, the term generallyrefers to a single oligopeptide which is optionally complexed with aradioisotope, but in certain instances may also refer tocomponents/portions of such compounds, intermediates used to synthesizesuch compounds, stereoisomers and/or optical isomers (including racemicmixtures) of disclosed compounds. The term compound shall include, whereapplicable, any and all relevant pharmaceutically acceptable saltsthereof.

The term “neutral amino acid” is an amino acid which has an unchargedsidechain at physiological pH. Neutral amino acids for use in thepresent invention include, for example, glycine, alanine, valine,leucine, isoleucine, norleucine, methionine, phenylalanine, serine,threonine and tyrosine. Preferred neutral amino acids include glycine,alanine, valine, leucine, isoleucine and norleucine. The term“positively charged amino acid” is an amino acid which has a positivelycharged sidechain at physiological pH. Preferred positively chargedamino acids for use in the present invention include arginine andlysine.

The term “radical” is used to describe a group which is covalentlybonded to another group in compounds according to the present invention.

The term “melanoma” is used to describe a malignant tumor of melanocyteswhich are found predominantly in skin but also in the bowel and the eye(see uveal melanoma), even though melanoma can be found in any part ofthe body. Melanoma is a form of cancer that begins in melanocytes, thecells that make skin pigment, or melanin. It may begin in a mole (skinmelanoma), but can also begin in other pigmented tissues. There areseveral types of melanoma, defined by where they first appear, includingskin and eye melanoma and in rare instances in the GI tract or lymphnodes

Melanoma is one of the rarer types of skin cancer but causes themajority of skin cancer related deaths. Malignant melanoma is a serioustype of skin cancer. It is due to uncontrolled growth of pigment cells,called melanocytes. Despite many years of intensive laboratory andclinical research, the sole effective cure is surgical resection of theprimary tumor before it achieves a Breslow thickness greater than 1 mm.

Around 160,000 new cases of melanoma are diagnosed worldwide each year.About 48,000 melanoma related deaths occur worldwide per year. Malignantmelanoma accounts for 75 percent of all deaths associated with skincancer. The treatment includes surgical removal of the tumor; adjuvanttreatment; chemo- and immunotherapy, or radiation therapy. The severityof melanoma is often characterized by the Clark level, which are forthin tumors and describe how deeply the cancer has spread into the skin,and the Breslow depth, which refers to the microscopic depth of tumorinvasion.

The following stages are identified in the progression of the melanomadisease state. Melanoma progresses from an early stage (in situ) throughan invasive stage, a high risk melanoma stage, a regional metastaticstage and a distant metastatic stage with varying degrees ofsurvivability, as set forth below.

Melanoma Stages: Stage 0: Melanoma in Situ (Clark Level I), 99.9%Survival Stage I/II: Invasive Melanoma, 85-95% Survival

T1a: Less than 1.00 mm primary, w/o Ulceration, Clark Level II-III

T1b: Less than 1.00 mm primary, w/o Ulceration or Clark Level IV-V

T2a: 1.00-2.00 mm primary, w/o Ulceration

Stage II: High Risk Melanoma, 40-85% Survival

T2b: 1.00-2.00 mm primary, w/o Ulceration

T3a: 2.00-4.00 mm primary, w/o Ulceration

T3b: 2.00-4.00 mm primary, w/o Ulceration

T4a: 4.00 mm or greater primary w/o Ulceration

T4b: 4.00 mm or greater primary w/o Ulceration

Stage III: Regional Metastasis, 25-60% Survival

N1: Single Positive Lymph Node

N2: 2-3 Positive Lymph Nodes OR Regional Skin/In-Transit Metastasis

N3: 4 Positive Lymph Nodes OR Lymph Node and Regional Skin/In TransitMetastases

Stage IV: Distant Metastasis, 9-15% Survival

M1a: Distant Skin Metastasis, Normal LDH

M1b: Lung Metastasis, Normal LDH

M1c: Other Distant Metastasis OR Any Distant Metastasis with ElevatedLDH

Based Upon AJCC 5-Year Survival with Proper Treatment

Tradition therapy of melanoma involves a number of treatment options.These generally include surgery, chemotherapy, radiation therapy andimmunotherapy (IL-2, other). In the case of surgery, treatment can varyand can include local excision, wide local excision, lymphadenectomy,sentinel lymph node biopsy and skin grafting. In the case ofchemotherapy, a standard chemotherapeutic agent dacarbazine (DTIC) isadministered to the patient in order to treat the cancer, generallythrough cancer cell death. In the case of radiation therapy, radiationis used as a palliative rather than a cure for melanoma. Radiationrelieves bone pain and other symptoms caused by metastases to the bones,brain, and organs such as the liver. Although not curative, radiationtreatment is being investigated for more widespread use in controllingother symptoms of skin cancer. In the case of immunotherapy (biologictreatment), a patient's natural immune system is raised or other immunecompositions (IL-2) are administered to the patient against the cancer.

“Metastatic melanoma” refers to a progressed form of melanoma whereinthe original cancer has metastasized to another area of the skin(regional or distant) or to other non-skin tissue (e.g., lungs, liver,brain, lymph system). Metastatic melanoma describes when melanoma hasspread into surrounding healthy tissue and through the bloodstream, orlymphatic system, to other parts of the body. If melanoma spreads tothese other areas, the cancer cells in the new tumor are still melanomacells but the disease is called metastatic melanoma.

Unlike early stages of melanoma, which can be treated successfully withearly diagnosis, the prognosis for patients diagnosed with metastaticmelanoma is poor, with survival rates of six to nine months. In the past35 years, the FDA has only approved two types of therapies formetastatic melanoma interleukin 2 (IL-2) and DTIC. The methods oftreatment for metastatic melanoma include radiation, immunotherapy,chemotherapy and palliative surgery. Currently, there are no approvedtherapies that significantly improve survival for patients withmetastatic melanoma.

The term “imaging”, “molecular imaging” or “radioimaging is used todescribe methods that use the nuclear properties of matter in diagnosisand therapy, pursuant to the present invention. More specifically, thepresent invention relies on molecular imaging because it produces imagesthat reflect biological processes that take place at the cellular andsubcellular level.

Molecular imaging is a discipline that unites molecular biology and invivo imaging. It enables the visualisation of the cellular function andthe follow-up of the molecular process in living organisms withoutperturbing them. The multiple and numerous potentialities of this fieldare applicable to the diagnosis and treatment of diseases such ascancer, in the present invention, in particular, melanoma, includingmetastatic melanoma. This technique also contributes to improving thetreatment of these disorders by optimizing the pre-clinical and clinicaltests of new medication. This approach also has a major economic impactdue to earlier and more precise diagnosis.

Molecular imaging differs from traditional imaging in that probeslabeled biomarkers are used to help image particular targets orpathways. Biomarkers interact chemically with their surroundings and inturn alter the image according to molecular changes occurring within thearea of interest. This process is markedly different from previousmethods of imaging which primarily imaged differences in qualities suchas density or water content. This ability to image fine molecularchanges opens up an incredible number of exciting possibilities formedical application, including early detection and treatment of disease,in particular, melanoma and metastatic melanoma according to the presentinvention.

There are a number of different imaging modalities that can be used fornoninvasive molecular imaging, using compounds according to the presentinvention. Each has different strengths and weaknesses and some are moreadept at imaging multiple targets or sites than others. This isimportant in instances where metastatic melanoma is suspected. Themodalities which can be used in the present invention are varied and inthe present invention principally include single photon emissioncomputed tomography (SPECT) and positron emission tomography (PET),discussed below.

The main purpose of SPECT when used in melanoma imaging pursuant to thepresent invention is to measure the distribution of radioisotope in skintissue, in particular, those skin regions and other tissues wheremelanoma, including metastatic melanoma, is suspected. The developmentof computed tomography in the 1970s allowed mapping of the distributionof the radioisotopes in tissue, and led to the technique now calledSPECT.

The imaging agent used in SPECT emits gamma rays, as opposed to thepositron emitters used in PET. There are a number of radioisotopes (suchas ^(99m)Tc, ¹¹¹In, ¹²³I, ²⁰¹Tl, ⁶⁷Ga, ^(99m)Tc and ²⁰³Pb, among othergamma ray emitters) that can be used in the present invention and imagedwith SPECT technology. In SPECT, where possible, by rotating the gammacamera around the area to be analysed, a three dimensional image of thedistribution of the radiotracer may be obtained by employing filteredback projection or other tomographic techniques. The radioisotopes usedin SPECT have relatively long half lives (a few hours to a few days)making them easy to produce and relatively cheap in comparison to otherradioisotopes. This represents the major advantage of SPECT as animaging technique, since it is significantly cheaper than PET or otherimaging methods such as magnetic resonance imaging (MRI). However, SPECTsometimes lacks exceptional spatial (i.e., where exactly the particleis) or temporal (i.e., did the contrast agent signal happen at aparticular millisecond or not) resolution.

Another imaging technique which finds particular use in the presentinvention is positron emission tomoaraphy (PET). In PET, a molecule istagged with a positron emitting isotope. These positrons (βparticles)interact with nearby electrons, emitting two 511,000 eV photons,directed 180 degrees apart in opposite directions. These photons arethen detected by the scanner which can estimate the density of positronannihilations in a specific area. When enough interactions andannihilations have occurred, the density of the original molecule may bemeasured in that area. Typical isotopes include ¹¹C, ¹³N, ¹⁵O, ¹⁸F,⁶⁴Cu, ⁶²Cu, ¹²⁴I, ⁷⁶Br, ⁸²Rb and ⁶⁸Ga, among others, including thepreferred ⁶⁶Ga, ⁶⁸Ga, ⁶⁴Cu, ⁸⁶Y. One of the major disadvantages of PETis that most of the radioisotopes must be made with a cyclotron, thusmaking the use of PET, in certain instances prohibitively expensive.Most of these probes also have a half life measured in minutes andhours, thus forcing the cyclotron, in many instances, to be on site.These factors can make PET sometimes prohibitively expensive, except incertain cases, which the present invention addresses in certain aspects.PET imaging does have many advantages though. First and foremost is itssensitivity: a typical PET scanner can detect between 10⁻¹¹ mol/L to10⁻¹² mol/L concentrations.

The term “effective” is used, to describe an amount of a compound,component or composition, which produces an intended effect when usedwithin the context of its use, which may be a diagnostic method, atherapeutic method, a method to monitor the progression of therapy orother method (chemical synthesis) pursuant to the present invention. Inthe case of therapeutic methods, an effective amount for treatingmelanoma, including metastatic melanoma, is that amount which shrinkscancerous tissue (e.g., tumor), produces a remission, prevents furthergrowth of the tumor and/or reduces the likelihood that the cancer in itsearly stages (in situ or invasive) does not progress further tometastatic melanoma. In preferred therapeutic aspects of the invention,the radioisotopes ¹⁸⁶Re and ¹⁸⁸Re are complexed to the cyclic peptidecompounds according to the present invention to produce unexpectedlyeffective therapeutic agents for the treatment of melanoma, includingmetastatic melanoma according to the present invention.

Noted here is that within the context of the use of the presentinvention, the patient will be receiving a radiation dose, whichprovides guidance to the amount of compound which is consideredeffective when used within the context of its use. A patient undergoinga nuclear medicine procedure will receive a radiation dose. Underpresent international guidelines it is assumed that any radiation dose,however small, presents a risk. The radiation doses delivered to apatient in a nuclear medicine investigation present a very small risk ofside effects, including inducing cancer in the patient. In this respectit is similar to the risk from X-ray investigations except that the doseis delivered internally rather than from an external source such as anX-ray machine.

The radiation dose from a diagnostic nuclear medicine procedure isexpressed as an effective dose with units of sieverts (usually given inmillisieverts, mSv). The effective dose resulting from an investigationis influenced by the amount of radioactivity administered inmegabecquerels (MBq), the physical properties of the radiopharmaceuticalused, its distribution in the body and its rate of clearance from thebody.

Effective doses can range from 6 μSv (0.006 mSv) for a 3 MBq chromium-51EDTA measurement of glomerular filtration rate to 37 mSv or more for a150 MBq thallium-201 non-specific tumour imaging procedure. The commonbone scan with 600 MBq of technetium-99m-MDP has an effective dose of 3mSv. Formerly, units of measurement were the Curie (Ci), being 3.7E10Bq, and also 1.0 grams of radium (Ra-226); the rad (radiation absorbeddose), now replaced by the Gray; and the rem (röntgen equivalent man),now replaced with the Sievert. The rad and rem are essentiallyequivalent for almost all nuclear medicine procedures, and only alpharadiation will produce a higher Rem or Sv value, due to its much higherrelative biological effectiveness (RBE).

The term “coadministration” or “combination therapy” is used to describea therapy in which at least two active compounds (one of which is acompound according to the present invention) in effective amounts areused to treat melanoma, including metastatic melanoma as otherwisedescribed herein at the same time. Although the term coadministrationpreferably includes the administration of two active compounds to thepatient at the same time, it is not necessary that the compounds beadministered to the patient at the same time, although effective amountsof the individual compounds will be present in the patient at the sametime. Compounds according to the present invention may be administeredwith one or more compounds including a chemotherapeutic agent such asdacarbazine (DTIC) or other anticancer agent useful in the treatment ofmelanoma and/or and immunotherapeutic agent such as IL-2 and/orα-interferon, among other compounds.

The term “treating” or “successfully treating” when used within thecontext of treating melanoma, including metastatic melanoma, shallinclude shrinking a tumor, curing melanoma, including melanoma which hasmetastasized (by causing a remission of the cancer in the patient) orreducing the likelihood or preventing the spread of the melanoma intoother organs. Melanoma, including metastatic melanoma, may be treatedusing compounds according to the present invention alone, or incombination with other methods and/or compounds including surgery,chemotherapy (especially the use of the chemotherapeutic agentdacarbazine or DTIC or a related anticancer agent), radiation therapy(i.e., with agents other than the present therapeutic compositions) andimmunotherapy (IL-2 and/or α-interferon).

In preferred aspects, R_(i) is selected from the group consisting of¹¹¹In, ⁸⁶Y, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ²⁰³Pb, ⁶⁴Cu and ^(99m)Tc when thecompounds are to be used diagnostically or to monitor therapeuticintervention and R_(I) is selected from the group consisting of ⁹⁰Y,¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Bi/²¹²Pb, ²¹³Bi, ¹⁴⁹Pm, ¹⁶⁶Ho and ¹⁵³Sm whencompounds according to the present invention are used in radiationtherapy to treat melanoma, including metastatic melanoma. Often,isotopes for use in therapy to treat melanoma, including metastaticmelanoma include ¹⁸⁶Re and ¹⁸⁸Re.

The present invention also relates to pharmaceutical compositionscomprising an effective amount of a compound for diagnostic and/ortherapeutic purposes in combination with a pharmaceutically acceptablecarrier, additive or excipient in pharmaceutical dosage form. Fordiagnostic purposes pharmaceutical compositions are formulated generallyin parenteral dosage form, especially for intravenous administration,although oral or topical formulations may be useful in certaininstances. In the case of the use of compounds according to the presentinvention for therapeutic purposes, the compositions are formulatedpreferably in parenteral or topical dosage forms, although orallyadministered dosage forms are also useful.

The compounds of the present invention, may, in accordance with theinvention, be administered in single or divided doses by oral,parenteral or topical routes. Administration of the active compound mayrange from a single intravenous injection to continuous (intravenousdrip) to several oral administrations per day (for example, Q.I.D.) andmay include oral, topical, parenteral, intramuscular, intravenous,sub-cutaneous, transdermal (which may include a penetration enhancementagent), buccal, sublingual and suppository administration, among otherroutes of administration. Enteric coated oral tablets may also be usedto enhance bioavailability of the compounds from an oral route ofadministration. The most effective dosage form will depend upon thepharmacokinetics of the particular agent chosen as well as the severityof disease in the patient. Administration of compounds according to thepresent invention as sprays, mists, or aerosols for intra-nasal,intra-tracheal or pulmonary administration may also be used. The presentinvention therefore also is directed to pharmaceutical compositionscomprising an effective amount of compound according to the presentinvention, optionally in combination with a pharmaceutically acceptablecarrier, additive or excipient.

The amount of compound used is that amount effective within the contextof the administration, whether that administration is for diagnosticpurposes or therapeutic purposes. A suitable oral dosage for a compoundaccording to the present invention would be in the range of about 0.01mg to 10 g or more per day, preferably about 0.1 mg to about 1 g perday. In parenteral formulations, a suitable dosage unit may contain from0.1 to 250 mg of said compounds, which may be administered from one tofour times per day (for diagnostic purpose, preferably once in a bolusdose), whereas for topical administration, formulations containing 0.01to 1% active ingredient are preferred. It should be understood, however,that the dosage administration from patient to patient will vary and thedosage for any particular patient will depend upon the clinician'sjudgment, who will use as criteria for fixing a proper dosage the sizeand condition of the patient as well as the patient's response to thedrug.

When the compounds of the present invention are to be administered bythe oral route, they may be administered as medicaments in the form ofpharmaceutical preparations which contain them in association with acompatible pharmaceutical carrier, additive or excipient material. Suchcarrier material can be an inert organic or inorganic carrier materialsuitable for oral administration. Examples of such carrier materials arewater, gelatin, talc, starch, magnesium stearate, gum arabic, vegetableoils, polyalkylene-glycols, petroleum jelly and the like.

The pharmaceutical preparations can be prepared in a conventional mannerand finished dosage forms can be solid dosage forms, for example,tablets, dragees, capsules, and the like, or liquid dosage forms, forexample solutions, suspensions, emulsions and the like.

The pharmaceutical preparations may be subjected to conventionalpharmaceutical operations such as sterilization. Further, thepharmaceutical preparations may contain conventional adjuvants such aspreservatives, stabilizers, emulsifiers, flavor-improvers, wettingagents, buffers, salts for varying the osmotic pressure and the like.Solid carrier material which can be used include, for example, starch,lactose mannitol, methyl cellulose, microcrystalline cellulose, talc,silica, dibasic calcium phosphate, and high molecular weight polymers(such as polyethylene glycol).

For parenteral use, a compound according to the present invention can beadministered in an aqueous or non-aqueous solution, suspension oremulsion in a pharmaceutically acceptable oil or a mixture of liquids,which may contain bacteriostatic agents, antioxidants, preservatives,buffers or other solutes to render the solution isotonic with the blood,thickening agents, suspending agents or other pharmaceuticallyacceptable additives. Additives of this type include, for example,tartrate, citrate and acetate buffers, ethanol, propylene glycol,polyethylene glycol, complex formers (such as EDTA), antioxidants (suchas sodium bisulfite, sodium metabisulfite, and ascorbic acid), highmolecular weight polymers (such as liquid polyethylene oxides) forviscosity regulation and polyethylene derivatives of sorbitolanhydrides. Preservatives may also be added if necessary, such asbenzoic acid, methyl or propyl paraben, benzalkonium chloride and otherquaternary ammonium compounds. In certain preferred diagnostic and/ortherapeutic embodiments, compounds according to the present inventionare administered intravenously in sterile saline solution.

The compounds of this invention may also be administered as solutionsfor nasal application and may contain in addition to the compounds ofthis invention suitable buffers, tonicity adjusters, microbialpreservatives, antioxidants and viscosity-increasing agents in anaqueous vehicle. Examples of agents used to increase viscosity arepolyvinyl alcohol, cellulose derivatives, polyvinylpyrrolidone,polysorbates or glycerin. Preservatives added may include benzalkoniumchloride, chloro-butanol or phenylethyl alcohol, among numerous others.

Additionally, the compounds provided by the invention can beadministered by suppository.

In certain aspects according to the present invention, where variouscancers are to be treated, the compounds may be co-administered with atleast one other anti-cancer agent such as dacarbazine (DTIC), amongothers, or an immunotherapeutic agent such as IL-2 and/or α-interferon.In addition, compounds according to the present invention may beadministered prior to, during or after surgery to remove melanomatissue.

Preparation of compounds according to the present invention proceedsusing standard synthetic chemical techniques which are readily availablein the art. Synthetic methods for obtaining compounds related to thepresent invention may be found in the examples section of the presentspecification. These methods can serve as guides for obtaining compoundsaccording to the present invention. In general, the present compoundsmay be made by routine chemical synthesis of peptides using methods wellknown in the art. One can readily follow the synthetic methods describedin the present specification with routine modification to provide all ofthe compounds described in the present application or using methodswhich are readily known in the art. The radionuclide is generallycomplexed to the oligopolypeptide after the oligo/polypeptide issynthesized, although alternative approaches to complexation can beused. Each of the two cyclic peptides (on the left- and right-side ofthe final compound) may be synthesized separately and then joined toeach other by condensing onto or with the linker molecule (depending onthe chemistry of the linker used). Cyclic peptide and/or the cyclicpeptide with no linker is synthesized using conventional peptidesynthesis (as otherwise described in the examples section or usingmethods readily available in the art using protecting group chemistry)and the various condensation and other reactions, etc. are readilyperformed using methods described herein or otherwise as readily knownin the art. Other approaches will be readily recognized to those ofordinary skill.

Once the compounds are synthesized, they may be formulated inpharmaceutical dosage form using conventional pharmaceutical formulationmethods readily available in the art by simply admixing compounds withchosen carriers, additives and/or excipients, depending upon the dosageform to be used and depending upon the use (diagnostic or therapeutic)of the compositions.

The following examples are provided to assist in describing the presentinvention. The details of these examples and the general description ofthe examples are for description purposes only and should be seen ortaken to limit the scope of the invention in any way.

EXAMPLES Chemicals and Reagents

Amino acid and resin were purchased from Advanced ChemTech Inc.(Louisville, Ky.) and Novabiochem (San Diego, Calif.). ¹²⁵I-Tyr²-[Nle⁴,DPhe⁷]-α-MSH {¹²⁵I-(Tyr²)-NDP-MSH} was obtained from PerkinElmer, Inc.(Shelton, Conn.) for MC1 receptor binding assay. ^(99m)TcO₄ ⁻ waspurchased from Cardinal Health (Albuquerque, N. Mex.) for peptideradiolabeling. All other chemicals used in this study were purchasedfrom Thermo Fisher Scientific (Waltham, Mass.) and used without furtherpurification. B16/F1 murine melanoma cells were obtained from AmericanType Culture Collection (Manassas, Va.).

MC1 Receptor Binding Affinity

The RAD-Lys-(Arg¹¹)CCMSH was synthesized according to our publishedprocedure [23], purified by reverse phase-high performance liquidchromatography (RP-HPLC) and characterized by LC-mass spectroscopy. TheIC₅₀ value of RAD-Lys-(Arg¹¹)CCMSH for the MC1 receptor was determinedin B16/F1 melanoma cells. Briefly, the B16/F1 cells were harvested andseeded into a 24-well cell culture plate (0.2×10⁶ cells/well) andincubated at 37° C. overnight. After being washed with binding medium{Modified Eagle's medium with 25 mMN-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid), pH 7.4, 0.2%bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}, the cells (n=3)were incubated at 25° C. for 2 h with approximately 30,000 counts perminute (cpm) of ¹²⁵I-(Tyr²) NDP-MSH in the presence of increasingconcentrations (10⁻¹³ to 10⁻⁶ M) of the peptide in 0.3 mL of bindingmedium. The reaction medium was aspirated after the incubation. Thecells were rinsed twice with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 Mphosphate buffered saline (PBS) and lysed in 0.5 mL of 1 N NaOH for 5min. The radioactivities associated with cells were measured in a Wallac1480 automated gamma counter (PerkinElmer, NJ). The IC₅₀ value ofRAD-Lys-(Arg¹¹)CCMSH was calculated using the Prism software (GraphPadSoftware, La Jolla, Calif.).

Cellular Internalization and Efflux of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH

RAD-Lys-(Arg¹¹)CCMSH was radiolabeled with ^(99m)Tc using the methoddescribed previously [23]. The radiolabeled peptide was purified tosingle species by Waters RP-HPLC (Milford, Mass.) on a Grace Vydac C-18reverse phase analytic column (Deerfield, Ill.) using a 20 min gradientof 16-26% acetonitrile in 20 mM HCl aqueous solution at a flow rate of 1mL/min. Cellular internalization and efflux of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH were evaluated in B16/F1 melanoma cells.Briefly, the B16/F1 cells in 24-well cell culture plates were incubatedat 25° C. for 20, 40, 60, 90 and 120 min (n=4) in the presence ofapproximately 200,000 cpm of HPLC purified^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH. After incubation, the reaction medium wasaspirated and the cells were rinsed twice with 0.5 mL of ice-cold pH7.4, 0.2% BSA/0.01 M PBS. Cellular internalization of^(99m)Tc-RAD-Lys-(Arg¹¹) CCMSH was assessed by washing the cells withacidic buffer [40 mM sodium acetate (pH 4.5) containing 0.9% NaCl and0.2% BSA] to remove the membrane-bound radioactivity. The remaininginternalized radioactivity was obtained by lysing the cells with 0.5 mLof 1 N NaOH for 5 min. Membrane-bound and internalized ^(99m)Tcactivities were counted in a gamma counter. Cellular efflux of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was determined by incubating the B16/F1cells with ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH for 2 h at 25° C., removingnon-specific-bound radioactivity with 2×0.5 mL of ice-cold pH 7.4, 0.2%BSA/0.01 M PBS rinse, and monitoring radioactivity released into cellculture medium. At time points of 20, 40, 60, 90 and 120 min, theradioactivities in medium, on cell surface and in cells were separatelycollected and counted in a gamma counter.

Biodistribution Studies

All the animal studies were conducted in compliance with InstitutionalAnimal Care and Use Committee approval. The biodistribution of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was determined in B16/F1 melanoma-bearingC57 mice (Harlan, Indianapolis, Ind.). C57 mice were subcutaneouslyinoculated on the right flank with 1×10⁶ B16/F1 cells. Tumor weightsreached approximately 0.2 g at 10 days post cell inoculation. Eachmelanoma-bearing mouse was injected with 0.037 MBq of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH via the tail vein. Groups of 5 mice weresacrificed at 0.5, 2, 4 and 24 h post-injection, and tumors and organsof interest were harvested, weighed and counted. Blood values were takenas 6.5% of the whole-body weight.

The specificity of tumor uptake was determined at 2 h post-injection byco-injecting ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH with 10 μg (6.1 nmol) ofunlabeled NDP-MSH. L-lysine co-injection is effective in decreasing therenal uptake of radiolabeled α-MSH peptides. To determine the effect ofL-lysine co-injection on the renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH, a group of 5 mice were injected with amixture of 0.037 MBq of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH and 12 mg ofL-lysine. The mice were sacrificed at 2 h post-injection, and tumors andorgans of interest were harvested, weighed and counted in a gammacounter.

Melanoma Imaging and Urinary Metabolites of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH

Approximately 6.7 MBq of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was injected in aB16/F1 melanoma-bearing C57 mouse for imaging and urine analysis. Themouse was euthanized at 2 h post-injection for small animal SPECT/CT(Nano-SPECT/CT®, Bioscan) imaging, as well as to collect urine foranalyzing the metabolites. The 9-min CT imaging was immediately followedby the whole-body SPECT scan. The SPECT scans of 24 projections wereacquired. Reconstructed SPECT and CT data were visualized andco-registered using InVivoScope (Bioscan, Washington D.C.). Thecollected urine sample was centrifuged at 16,000 g for 5 min before theHPLC analysis. Thereafter, aliquots of the urine were injected into theHPLC. A 20-minute gradient of 16-26% acetonitrile/20 mM HCl with a flowrate of 1 mL/min was used for urine analysis.

Statistical Methods

Statistical analysis was performed using the Student's t-test forunpaired data to determine the significance of differences in tumor andkidney uptake between the groups in the biodistribution studieswith/without peptide blockade or with/without L-lysine co-injection.Differences at the 95% confidence level (p<0.05) were consideredsignificant.

Results

RAD-Lys-(Arg¹¹)CCMSH was synthesized, purified by RP-HPLC andcharacterized by electrospray ionization mass spectrometry. Schematicstructure of RAD-Lys-(Arg¹¹)CCMSH is presented in FIG. 1. The structureof RGD-Lys-(Arg¹¹)CCMSH was cited from ref. 23 for comparison. Thecompetitive binding curve of RAD-Lys-(Arg¹¹)CCMSH is presented in FIG.2. The IC₅₀ value of RAD-Lys-(Arg¹¹)CCMSH was 0.26 nM in B16/F1 melanomacells. The peptide was readily labeled with ^(99m)Tc with greater than90% radiolabeling yield. ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was completelyseparated from its excess non-labeled peptide by RP-HPLC. The retentiontimes of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH and RAD-Lys-(Arg¹¹)CCMSH was 12.6and 10.7 min, respectively. ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH showed greaterthan 99% radiochemical purity after HPLC purification.

Cellular internalization and efflux of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH inB16/F1 melanoma cells are presented in FIG. 3.^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH exhibited rapid cellular internalizationand extended cellular retention. There was 59.20±5.10% of the^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH activity internalized at 20 min postincubation. There was 78.24±1.13% of the ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHactivity internalized after 2 h incubation. The efflux resultsdemonstrated that 79.44±3.61% of the ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSHactivity remained inside the cells 2 h after incubating cells in culturemedium.

The melanoma targeting and pharmacokinetic properties of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearingC57 mice. The biodistribution results of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHare shown in Table 1, FIG. 7. ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH exhibitedrapid and high tumor uptake in melanoma-bearing mice. The tumor uptakewas 16.65±1.91% ID/g at 0.5 h post-injection.^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH reached its peak tumor uptake of19.91±4.02% ID/g at 2 h post-injection. There was 18.01±3.51% ID/g ofthe ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH activity remained in tumor at 4 hpost-injection. The tumor uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHgradually decreased to 9.24±3.63% ID/g 24 h post-injection. In peptideblocking study, the tumor uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH with10 μg (6.1 nmol) of non-radiolabeled NDP-MSH co-injection was only 7.8%of the tumor uptake without NDP-MSH co-injection at 2 h post-injection(p<0.01), demonstrating that the tumor uptake was specific and MC1receptor-mediated. Whole-body clearance of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSHwas rapid, with approximately 63% of the injected radioactivity clearedthrough the urinary system by 2 h post-injection (FIG. 7, Table 1)Normal organ uptake of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH was generally low(<2.2% ID/g) except for kidneys after 2 h post-injection. Hightumor/blood and tumor/muscle uptake ratios were demonstrated as early as0.5 h post-injection (Figure, Table 1). The renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH reached its peak value of 127.41±17.32%ID/g at 0.5 h post-injection. The renal uptake decreased to 33.19±3.39%ID/g at 24 h post-injection. L-lysine co-injection significantly(p<0.05) reduced the renal uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH to50.1±18.56% ID/g at 2 h post-injection without affecting the tumoruptake.

Melanoma imaging property of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was examinedin a B16/F1 melanoma-bearing C57 mouse. The whole-body SPECT/CT image ispresented in FIG. 4. Flank melanoma tumors were visualized clearly by^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH at 2 h post-injection.^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH exhibited high tumor to normal organuptake ratios except for kidney. The urine collected from the imagingmouse was analyzed for the metabolites by HPLC. The urinary HPLC profileof ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH is shown in FIG. 5. Approximately 82%of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH remained intact, whereas 18% of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was transformed to a more lipophilicmetabolite at 2 h post-injection.

DISCUSSION

The inventors successfully developed a novel^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH hybrid peptide to target both MC1 andα_(v)β₃ integrin receptors for M21 human melanoma imaging [23]. Both MC1and α_(v)β₃ integrin receptors are over-expressed in M21 melanoma cells[23], making it a suitable melanoma cell line for evaluating dualreceptor-targeting ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH hybrid peptide. It isknown that the switch from the RGD to RAD sacrifices the bindingaffinity of the RGD to the α_(v)β₃ integrin receptor. Not surprisingly,the switch from RGD to RAD in the hybrid peptide decreased the α_(v)β₃integrin receptor binding affinity of RAD-Lys-(Arg¹¹)CCMSH by 248-foldin M21 melanoma cells. Surprisingly, the inventors found that the switchfrom RGD to RAD in the hybrid peptide increased the MC1 receptor bindingaffinity of RAD-Lys-(Arg¹¹)CCMSH by 6.7-fold in M21 melanoma cells [23].To investigate whether such change in MC1 receptor binding affinitycould lead to enhanced melanoma uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHcompared to ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH, we evaluated the receptorbinding and melanoma targeting properties of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH in B16/F1 melanoma cells andmelanoma-bearing mice in this study. ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSHtargeted both MC1 and α_(v)β₃ integrin receptors, whereas^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH only targeted the MC1 receptors. We choseB16/F1 melanoma cells for this study because only MC1 receptors (ratherthan α_(v)β₃ integrin receptors) are over-expressed on B16/F1 cells[24]. Thus, selection of B16/F1 melanoma cells could minimize thecontribution of α_(v)β₃ integrin receptors to the melanoma uptake ofdual receptor-targeting ^(99m) Tc-RGD-Lys-(Arg¹¹)CCMSH.

The structural difference between RAD-Lys-(Arg¹¹)CCMSH andRGD-Lys-(Arg¹¹)CCMSH was one more methyl group in Ala compared to Gly(FIG. 1). Despite such slight difference in structure,RAD-Lys-(Arg¹¹)CCMSH displayed much stronger MC1 receptor bindingaffinity than RGD-Lys-(Arg¹¹)CCMSH in B16/F1 melanoma cells. The MC1receptor binding affinity of RAD-Lys-(Arg¹¹)CCMSH andRAD-Lys-(Arg¹¹)CCMSH was 0.26 and 2.1 nM in B16/F1 cells [24]. Although^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH displayed similar rapid internalizationand extended retention pattern as ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH, more^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH activity was internalized and remained inB16/F1 melanoma cells.

^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH displayed a rapid B16/F1 melanoma uptakeof 11.06±1.41% ID/g at 0.5 h post-injection and reached its peak tumoruptake of 14.83±2.94% ID/g at 2 h post-injection in our previous report[24]. The tumor uptake of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH was 12.57±2.53and 7.59±2.04% ID/g at 4 h and 24 h post-injection, respectively [24].In this study, the switch from RGD to RAD significantly (p<0.05)improved the tumor uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH at 0.5, 2 and4 h post-injection. The tumor uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSHwas 1.51, 1.34 and 1.43 times the tumor uptake of^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH at 0.5, 2 and 4 h post-injection (FIG.6A), respectively. The improved melanoma uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was likely due to its stronger MC1receptor binding affinity compared to ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH(0.26 vs. 2.1 nM). Meanwhile, the replacement of the RGD motif with RADsignificantly (p<0.05) decreased the liver uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH at 0.5, 4 and 24 h post-injection. Theliver uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was 66.2, 61.9 and 72.3%of the liver uptake of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH at 0.5, 2 and 24 hpost-injection (FIG. 6B), respectively.

B16/F1 melanoma lesions were clearly visualized by SPECT/CT imagingusing ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH as an imaging probe 2 h postinjection, highlighting the potential use of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH for melanoma imaging.^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH displayed high tumor to normal organuptake ratios except for kidney, which was coincident with thebiodistribution results (Table 1). As shown in FIG. 6C,^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH exhibited significantly higher renaluptake than that of ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH at 0.5, 2 and 4 hpost-injection. The renal uptake of ^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was1.84, 1.39 and 1.42 times the renal uptake of^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSH at 0.5, 2 and 4 h post-injection,respectively. Co-injection of peptide blockade did not reduce the renaluptake, indicating that the renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH was not MC1 receptor-mediated. L-lysineco-injection significantly decreased the renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH by 46%, demonstrating that L-lysineco-injection could be used to reduce the renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH. Importantly, the success of L-lysineco-injection suggested that the positive charge played a key role in therenal uptake of ^(99m)Tc-RAD-Lys-(Are)CCMSH. It was worthwhile to notethat the epsilon amino group of Lys linking the RAD motif and CCMSHcontributed a positive charge to the overall charge of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH. Reduction of the overall positive chargeof radiolabeled α-MSH peptide via structural modification has beensuccessfully utilized to decrease the renal uptake by 50% [11].Accordingly, it would be likely to decrease the renal uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH by substituting the Lys linker withneutral amino acid or polyethylene glycol (PEG) linker in our futurestudies.

CONCLUSIONS

The switch from RGD to RAD significantly enhanced the melanoma uptake of^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH compared to ^(99m)Tc-RGD-Lys-(Arg¹¹)CCMSHin B16/F1 melanoma-bearing C57 mice. B16/F1 melanoma lesions wereclearly visualized by SPECT/CT imaging using^(99m)Tc-RAD-Lys-(Arg¹¹)CCMSH as an imaging probe, highlighting itspotential use as an imaging probe for melanoma detection as well astherapeutics.

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1. A compound according to the chemical structure:

Where Q is an amino acid unit selected from the group consisting ofglutamic acid and aspartic acid; R is an amino acid unit selected fromthe group consisting of valine, threonine, leucine, and isoleucine; V isan amino acid residue selected from the group consisting of asparticacid and glutamic acid; W is an amino acid selected from the groupconsisting of aspartic acid and glutamic acid; X is an amino acidresidue selected from the group consisting of alanine, valine,threonine, leucine, isoleucine, serine, aspartic acid and glutamic acid;Y is an amino acid residue selected from the group consisting ofarginine, lysine, alanine, valine, threonine, leucine, isoleucine,serine, aspartic acid and glutamic acid; L is absent, a single aminoacid selected from the group consisting of glycine, alanine, β-alanine,lysine and arginine, or a linker group according to the formula:

Where each X¹ is independently an amino acid residue (preferably, forexample, an amino acid group which is neutral (e.g. a neutral amino acidsuch as norleucine (Nle), leucine, isoleucine, glycine or alanine) or ispositively charged at physiological pH (arginine, lysine) and ispreferably selected from the group consisting of glycine, alanine,arginine or lysine, or is an amino acid linker comprising an alkylenegroup which is optionally substituted with one or more C₁-C₃ alkyl orC₁-C₃ alkanol group(s) or an ethylene glycol containing group accordingto the chemical structures:

Where ABC is an amino acid linker wherein A is absent or is a neutral orpositively charged amino acid at physiological pH; B is a neutral orpositively charged amino acid at physiological pH; C is absent or is aneutral or negatively charged amino acid at physiological pH; m is aninteger from 0 to 250; Each n is independently 0 to 10; p is an integerfrom 0 to 20; k is an integer from 0 to 10; i is an integer from 0 to10; s is an integer from 0 to 10; and M is a radioisotope, or apharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1 wherein said radioisotope is a polyvalent cationic radioisotope,even more preferably selected from the group consisting of ⁸⁶Y, ⁹⁰Y,¹¹¹In, ¹⁷⁷Lu, ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁶⁴Cu, ⁶⁷Cu, ⁷¹As,⁷⁶As, ⁷⁷As, ⁷²As, ⁷⁶As, ⁷⁷As, ⁶⁵Zn, ⁴⁸V, ²⁰³Pb, ²⁰⁹Pb, ²¹²Pb, ¹⁶⁶Ho,¹⁴⁹Pm, ¹⁵³Sm, ²⁰¹Tl, ¹⁸⁸Re, ¹⁸⁶Re, and ^(99m)Tc.
 3. The compoundaccording to claim 1 wherein X is alanine.
 4. The compound according toclaim 1 wherein said compound is

Where Q is glutamic acid; R is valine; V is aspartic acid; W is asparticacid; X is alanine, valine, threonine, leucine or isoleucine; Y isarginine or lysine; L is a single amino acid selected from the groupconsisting of glycine, alanine, β-alanine, valine, leucine, isoleucine,lysine and arginine, or a

group, Where p is an integer 0-6; k is an integer from 0 to 10; i is aninteger from 0 to 10; s is an integer from 0 to 10; and M is ^(99m)Tc,¹⁸⁸Re or ¹⁸⁶Re a pharmaceutically acceptable salt thereof.
 5. Thecompound according to claim 1 wherein L is

and p is 1-6.
 6. The compound according to claim 1 wherein L is

s and i are 0; and k is 1-5.
 7. The compound according to claim 1wherein M is ^(99m)Tc, ¹⁸⁸Re or ¹⁸⁶Re.
 8. (canceled)
 9. The compoundaccording to claim 1 wherein L is selected from the group consisting oflysine and arginine.
 10. (canceled)
 11. A method of diagnosing melanomain a patient comprising exposing a patient suspected of having melanomawith a compound according to claim 1 and determining whether saidcompound has bound to tissue in said patient in an amount whichevidences the existence of a melanoma tumor.
 12. (canceled)
 13. Themethod according to claim 11 wherein said compound is according to claim2.
 14. The method according to claim 11 wherein said compound isaccording to claim
 3. 15. A method of treating melanoma in a patient inneed thereof comprising administering to said patient an effectiveamount of a compound according to claim
 1. 16. (canceled)
 17. (canceled)18. (canceled)
 19. The method according to claim 15 wherein saidcompound is administered in topical dosage form.
 20. The methodaccording to claim 19 wherein said compound is administered directlyonto melanoma tissue on the skin.
 21. The method according to claim 15wherein said compound is administered in parenteral dosage form. 22.(canceled)
 23. (canceled)
 24. The method according to claim 15 whereinsaid melanoma is metastatic melanoma.
 25. A pharmaceutical compositioncomprising an effective amount of a compound according to claim 1 incombination with a pharmaceutically acceptable carrier, additive orexcipient.
 26. (canceled)
 27. The composition according to claim 25 intopical dosage form.
 28. The composition according to claim 25 inparenteral dosage form.
 29. (canceled)
 30. (canceled)
 31. A method ofmonitoring melanoma therapy in a patient undergoing such therapycomprising administering a compound according to claim 1 to saidpatient, and diagnosing the extent of melanoma in said patient overtime, wherein a decrease in melanoma tissue over a period of treatmentis evidence of success of said treatment.
 32. (canceled)